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S A Bernhard

Publications and source records attributed to S A Bernhard.

At least 37 records · Page 2Linked to original sources
PubMed1981

Transient and steady-state kinetic studies of sodium-potassium adenosine triphosphatase using beta-(2-furyl)acryloyl phosphate as chromophoric substrate assay.

A convenient and highly specific continuous spectrophotometric assay for sodium-potassium adenosine triphosphate activity utilizing the rapidly hydrolyzed and high-affinity chromophoric substrate beta-(2-furyl)acryloyl phosphate (FAP) is described. The Na/K-ATPase-catalyzed hydrolysis of FAP is faster than that for ATP under all ionic conditions. The rate is neither inhibited nor activated by Na+; it is dependent on [K+] and on [Mg2+]. The hydrolysis of FAP to furylacrylate is accompanied by a large shift in the UV absorbance maximum. The spectrum of FAP, but not furylacrylate, is sensitive to noncovalent ligation with Mg2+, a happenstance which permits the identification of Mg2+FAP, and consequently allows for a probe of the role of Mg2+ in the catalysis. Mg2+ binding to the active site is essential for catalysis. MgFAP is more tightly bound to the site than is FAP2-, but the complex is not obligatory for catalysis. The formation of a phosphoryl-enzyme intermediate is not evident in the reaction of FAP with the enzyme. Transient kinetic experiments, utilizing an excess of MgFAP, demonstrate a unique steady-state rate-limiting production of furylacrylate. These results indicate that the pathway demonstrated with ATP is not appropriate to the FAPase mechanism. The results suggest that acyl phosphates are good "phosphatase" substrates either because they are analogues of the phosphatase-specific phosphoryl-enzyme or because they react exclusively with the isomerized "E2" form of the enzyme.

Acrylates↗
PubMed1981

Structural and functional consequences of subunit interactions in glyceraldehyde 3-phosphate dehydrogenase.

A variety of species of GPDH undergo acylation at two of the four active cystein sites per mole of tetrameric enzyme. This reaction requires tightly bound NAD+, a situation restricted to two of the four NAD sites per tetramer. S leads to N acyl transfer from cysteins to lysine in the diacyl enzyme yields an inactive enzyme. The thiol ester bond of acyl enzyme is activated by NAD+ and NADH for the group transfer and reduction reactions, respectively. In furyl acryloyl-GPDH this activation is accompanied by large acyl-spectral shifts, a "blue shift" with NADH and a "red shift" with NAD+. The group transfer reaction as well as spectral shifts show biphasic kinetics. The amplitude of the fast phase of NAD+-induced spectral change in apo-enzyme is equal to that of the fast phase in phosphorolysis (or arsenolysis) at low [NAD+]. The kinetic pattern of spectral shifts by NAD+ and NADH are complementary; the amplitude of the fast phase in one is equal to that of the slow phase in the other. It has been proposed that the acyl enzyme exists in two conformational states. The relative proportion of these states varies with the extent of covalent (acyl group) or non-covalent (NAD+ or NADH) ligation in a manner consistent with the allosteric model of Monod, Wyman and Changeux. These conclusions apply equally to the true substrate acyl enzyme. With 1,3-diphosphoglycerate, a tetra-acylated enzyme is obtained. Two of these four acyl groups react very much faster than the remaining two. A comparison of their specific rates with the steady state turnover numbers indicates that only the less reactive two acyl groups govern the turnover number of the enzyme.

Binding Sites↗
PubMed1980

Major component of acetylcholinesterase in Torpedo electroplax is not basal lamina associated.

Electroplax tissue from Torpedo californica contains two major structural forms of the enzyme acetylcholinesterase. One form, composed of tetrameric protomers which are further aggregated by interactions among associated collagenous "tail fibers", has been well characterized previously. This form is associated in situ with the basal lamina. The other form is described and characterized herein. This latter form accounts for at least 50% of the acetylcholinesterase activity of the tissue. This enzyme associated with the tissue phospholipids. It aggregates in aqueous solution but readily dissociates to dimers in 1% sodium cholate solution, a solvent in which it is both soluble and catalytically fully active. The same dimer is obtained in sodium dodecyl sulfate solution where the enzyme is denatured. Denaturation in the presence of the reductant dithiothreitol results in the formation of a single 80000-dalton subunit. The purified enzyme contains no collagenous component. It is not derivable from the collagenous "tailed-enzyme" form in the tissue homogenate. However, the two enzymes have similar molecular weight catalytic subunits and the same substrate-dependent turnover numbers (per active site) for a variety of choline esters which are generally utilized to distinguish specific esterase function. In the tissue homogenate each form of the enzyme is associated with a characteristic structural component (phospholipid or collagen). By implication, acetylcholinesterase function is localized in situ in the phospholipid membrane as well as at the basal lamina.

Acetylcholinesterase↗
PubMed1973

Activation of a covalent enzyme-substrate bond by noncovalent interaction with an effector.

The absorption spectrum of an activesite specific chromophoric acyl enzyme, sturgeon beta-(2-furyl)-acryloyl-glyceraldehyde-3-phosphate dehydrogenase, is reported. This acyl enzyme undergoes all of the catalyzed reactions characteristic of the intermediate of the physiological acyl enzyme, 3-phospho-D-glyceroyl-glyceraldehyde-3-phosphate dehydrogenease. The rates of reactions of both these acyl enzymes depend strongly on the extent of interaction of the acyl enzyme with the oxidized coenzyme, NAD(+), even where the "redox" properties of the coenzyme are not required. Likewise, the spectral properties of chromophoric acyl enzyme are affected by the extent of bound NAD. Under the pseudophysiological conditions reported herein, there is a stoichiometric limitation of two furylacryloyl-acyl groups per enzyme molecule containing four covalently-equivalent subunits. The binding of NAD both to the apoenzyme and to the diacyl enzyme is heterogeneous: at low extents of NAD occupancy, NAD binding is stronger. The binding to acyl enzyme can be quantitatively described by an enzyme model involving a tetramer with 2-fold symmetry, and consequently containing equal numbers of two classes of sites. NAD binding to difurylacryloyl-enzyme occurs virtually discretely, first to the two unmodified (tight-binding) sites, followed by looser binding to the two acyl-sites. NAD occupancy at these latter sites transforms the chromophoric acyl spectrum from that characteristic of a model furylacryloyl-thiol ester in H(2)O to a highly perturbed furylacryloyl spectrum characteristic of monomeric native "active-thiol" furylacryloyl-enzymes. Likewise the acyl reactivity towards arsenolysis depends on the extent of NAD bound to the loose sites. Elimination of the tight binding of NAD to the difurylacryloyl enzyme tetramer by alkylation of the remaining two free SH groups with iodoacetate has no apparent influence on the NAD-dependent furylacryloyl-spectral perturbation at the "two equivalent acyl sites," even though it eliminates the apparent "negative cooperativity" in NAD binding.

Acrylates↗